Adjuvants are sometimes used to improve the immune response raised to any given antigen. However the inclusion of adjuvants into a vaccine or immunogenic composition increases the complexity of preparation of the components as well as the complexity of distribution and formulation of the vaccine composition. The preparation of each of the adjuvant components as well as the antigenic component must be considered by formulators. This is particularly true because for example the pH of adjuvant components in solution may be very different from the optimal pH for a given antigen and these differences need to be carefully controlled and managed to prevent, for example precipation or loss of desirable properties of the components. The pH of the antigen in water for injection may, for example be about pH7 or slightly higher and when the adjuvant is added the pH may be as low as pH6.3. The antigen may, for example not be stable when stored for prolonged periods at this pH.
The components must then be formulated and distributed in a form that is as stable as possible because pharmaceutical products for human use must be well characterized, stable and safe before they can be approved for marketing. For this reason long term stability studies must be performed on the final formulation to ensure that it meets the relevant criteria. The information generated in such long term studies is used to support submission to regulatory authorities such as the FDA (Federal Drugs Authority—the body responsible for approving medicines in the USA) to show the product is suitable for use in humans.
Freeze-drying or lyophilisation, is used generally to increase the stability and hence storage life of material including pharmaceutical materials such as an antigen used in vaccines.
Often lyophilised antigenic compositions are provided to health care professions for reconstitution with diluent (for example water for injection [WFI] or in some instances a liquid adjuvant formulation) shortly before administration to the patient. In this way the period of time that the various components of the final vaccine are maintained in close proximity is minimised.
Many factors must be considered when antigens are lyophilised to form lyo cakes (the dry product from lyophilisation). For example, the antigenicity/immunogenicity of the antigen should be maintained in lyophilised form. The antigen must not aggregate or degrade whilst in lyophilised form. The lyo cake must be well formed and not collapse. Finally, the antigen must of course be in a form which dissolves rapidly when reconstituted. Where the solution for reconstitution is not simply WFI, for example when the antigen is reconstituted with liquid adjuvant, then the impact of the components of the solution on the properties of the reconstituted product needs to be considered.
As mentioned adjuvants have been used for many years to improve the immune response to the antigenic component of a vaccine. A particularly potent adjuvant combination is one comprising 3Deacylated-Monophosphoryl Lipid A (3D-MPL) and a saponin, particularly QS21, a purified fraction of saponin extracted from the bark of Quillaja saponaria Monara. This combination can be provided, for example as an oil in water emulsion, liposomal formulation or the like.
In previous clinical trials with antigens, for example with malaria antigens such as RTS,S the lyophilized antigen is provided and a separate vial of liquid adjuvant, for example an oil in water formulation of MPL and QS21 or a liposomal formulation of MPL and QS21 for reconstituting the antigen is also provided. The individual components are combined to form the final vaccine composition shortly before administration.
Certain immunostimulatory oligonucleotides containing unmethylated CpG dinucleotides (“CpG”) are TLR9 ligands and have been identified as being adjuvants when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al., J. Immunol, 1998, 160(2):870-876; McCluskie and Davis, J. Immunol., 1998, 161(9):4463-6). CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in DNA. Historically, it was observed that the DNA fraction of BCG could exert an anti-tumour effect. In further studies, synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity. The central role of the CG motif in immunostimulation was later elucidated in a publication by Krieg, Nature 374, p 546 1995. Detailed analysis has shown that the CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA. The immunostimulatory sequence is often: Purine, Purine, C, G, pyrimidine, pyrimidine; wherein the dinucleotide CG motif is not methylated, but other unmethylated CpG sequences are known to be immunostimulatory and may be used in the present invention.
It has also been shown that an immunostimulatory oligonucleotide can retain immunological activity when the Guanosine is mutated to a 7-deazaguanosine motif (WO 03057822).
These immunostimulatory oligonucleotides are thought to have an acidic pH in solution, for example below pH 7, such as 6.3, 6.1 or lower. This may make them difficult to incorporate in liquid vaccine formulations because they are dissimilar to other components in the formulations. As discussed this may cause precipitation and/or long term stability problems.
It is thought that these immunostimulatory oligonucleotides are likely to be very effective adjuvants, particularly when used in combination with existing adjuvant combinations such as 3D-MPL and QS21. It is expected that such adjuvants will be employed in diseases that have so far been difficult to provide effective vaccines for, such as HIV, cancer and possibly malaria.
There are a number of different ways in which adjuvants can be included in vaccines, but they must be included in a way which does not affect the stability either of themselves or the antigenic composition and also in a way which will not place an undue burden on the healthcare professional reconstituting the vaccine. The simplest way to achieve this would be to put additional components into additional vials such that they would be kept separate until just before reconstitution, thereby minimising the time during which the components could affect each other. This means the antigen and the immunostimulatory oligonucleotide would each be provided in a separate vials. Then if further adjuvant components such as MPL and QS21 are employed these can be provided as a liquid mixture in a third vial. However, an increasing number of components in an increasing number of vials leads to increased costs, waste and importantly to an increase in the possibility of mistakes during constitution.